ES2914053T3 - Reinforced engineered biomaterials and manufacturing methods thereof - Google Patents

Abstract

Fibre-reinforced biological tissue composite material, the material comprising: a fibrous support comprising a plurality of fibers, wherein the fibers are surrounded by extracellular matrix, and wherein the extracellular matrix and the plurality of fibers are cross-linked to each other .

Description

DESCRIPTION

Reinforced engineered biomaterials and manufacturing methods thereof

Countryside

Described herein are methods of manufacturing engineered leather incorporating a reinforcing structure, as well as the resulting leather formed by these methods. These engineered leathers can be used in place of, or in combination with, animal hide leathers.

Background

Leather is used in a wide variety of applications, including furniture upholstery, clothing, shoes, luggage, bags and accessories, and automotive applications. Currently, animal skins are used as raw material for natural leather. However, cattle hides raise environmental concerns because raising cattle requires enormous amounts of feed, pasture, water, and fossil fuel. Cattle also produce significant pollution to the air and waterways. Furthermore, the use of animal skins to produce leather is objectionable to people with a social conscience. The global leather industry slaughters more than a billion animals a year. Most leather comes from countries with no animal welfare laws or with laws that are largely or not fully enforced. Leather produced without killing animals would be a great novelty and would have fashion appeal.

Although synthetic leather was developed to address some of these concerns, it lacks the quality, durability, and prestige of natural leather. Until now, no scientifically sound and industrially viable procedures have been developed to produce natural leather. Accordingly, there is a need for a solution to the demands for alternatives to leather produced from live animals. There is a need for alternatives to leather produced from live animals and, in particular, there is a need for a leather material that is durable and can be produced sustainably.

Natural leather is typically a durable, flexible material created by tanning rawhide and animal hides, often cattle hides. Tanning is generally understood to be the process of treating animal hides to produce leather. Tanning can be carried out in a number of well-known ways, including vegetable tanning (for example, using tannin), chrome tanning (chromium salts, including chrome sulfate), aldehyde tanning (using oxazolidine or glutaraldehyde compounds), syntans (tannins synthetics, using aromatic polymers) and the like.

Natural leather is normally prepared in three main parts: preparatory phases, tanning and semi-finished leather formation. Surface coating may also be included. The preparatory steps prepare the hide/hide for tanning, and unwanted components are removed from the raw hide. Preparatory steps may include: holding, soaking (rehydration), liming, unliming, fleshing (removal of subcutaneous material), splitting, reliming, deliming (to remove liming and liming chemicals), rendering (protein proteolysis) , degreasing, pilling, bleaching, pickling (pH change), acid and salt removal ( de-pickling), etc.

Tanning is done to convert the proteins in the hide/skin into a stable material that will not rot, while allowing the material to remain flexible. Chrome is the most frequently used tanning material. The pH of the hide/hide can be adjusted (eg lowered, eg down to pH 2.8-3.2) to improve tannability; after tanning, the pH can be raised ("basification" to a slightly higher level, eg pH 3.8-4.2).

The formation of semi-finished leather refers to the treatment after tanning which may include coloring (dyeing), thinning, drying or moisturizing and the like. Examples of semi-finished leather-forming techniques include: wetting (rehydration), shortening (drying), splitting (into thinner layers), shaving, neutralizing (adjusting the pH to a more neutral level), retanning, dyeing, oiling, filling , filling, peeling, whitening, fixing unbonded chemicals, hardening, conditioning, softening, polishing, etc.

In practice, the process for converting animal skin into leather may include sequential steps such as: dehairing/hairing, liming, deliming and rendering, pickling, tanning, neutralizing/dyeing and oiling, drying and finishing. The unhairing process can chemically remove the hair (for example, using an alkaline solution), while the lime step (for example, using an alkaline and sulfur solution) can further complete the hair removal and swelling process (" open”) the collagen. During tanning, the structure of the hide can be stabilized in the "open" form by replacing some of the collagen with complex chromium ions. Depending on the compounds used, the color and texture of the leather can change. Tanned leather can be much more flexible than raw hide and also more durable.

Skin, or animal hide, is made up primarily of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 different types of collagen; animal skin is normally type 1 collagen (thus the term collagen is normally assumed to be type 1 collagen), although other types of collagen may be used for leather formation. Collagens are characterized by a repeating triplet of amino acids, -(Gly-XY)„-, such that approximately one-third of the amino acid residues found in collagen are glycine. X is often proline and Y is often hydroxyproline. Therefore, the structure of collagen may consist of interlocking triple units of peptide chains of different lengths. Different animals can produce different amino acid compositions of collagen, which can result in different properties (and differences in the resulting leather). Collagen fiber monomers can be produced from alpha chains approximately 1050 amino acids long, so that the triple helix takes the form of a rod approximately 300 nm long, with a diameter of 1.5 nm. In the production of the extracellular matrix by fibroblast skin cells, triple-stranded monomers can be synthesized and the monomers can bind together to form a fibrous form. These triple helices can be held together by salt bonds, hydrogen bonds, hydrophobic bonds, and covalent bonds. Triple helices can join together in bundles called fibrils, and bundles of fibrils join together to create fibers. The fibers normally divide and join together along a layer of skin. Variations in crosslinking or bonding can provide strength to the material. The fibers can have a variety of diameters. In addition to type I collagen, the skin (skin) may also include other types of collagen, including type III collagen (reticulin), type IV collagen, and type VII collagen.

Previous attempts to obtain engineered leathers have been unsuccessful or impractical. For example, EP 1589098 ("the '098 application") describes a method of growing seeded fibroblasts on three-dimensional bioactive supports. The supports may be composed of collagen waste material from a tanning ("splitting") process, pure collagen microparticles, collagen waste material particles, or synthetic supports (eg, composed of polymers such as HYAFF). The addition of the support material complicates and adds to the cost of his proposed process and also affects the properties of any leather produced in this way.

The '098 application is an example of a support technique for culturing leather, however, a method such as this, using a support of engineered or scrap collagen materials, has not been widely used because it is expensive and difficult to manufacture. work, and has proven to be technically difficult to work with and market.

To date, the manufacture of leather by ex vivo techniques (eg cell culture) has proven difficult. One potential advantage of cultured (i.e., engineered) leather is the ability to add materials, including synthetic materials, during the manufacturing process, so that such materials can be integrated into the resulting leather, for example, to provide added strength. , durability and functionality. Although references such as US Patent Publication No. 2012/0023777 to Greene have suggested cultivating leather by adding elements to the cultivated leather, such as reinforcing supports or spherical members, including hollow members, which can modify the density, weight and the stiffness of the resulting material. Unfortunately, references such as this one do not adequately describe how such materials can be made, particularly in terms of quantities and sizes necessary for significant commercial manufacture.

Described herein are engineered leathers that may include one or more reinforcing materials, that can be processed in a simple and highly robust manner that can be scaled so that commercially relevant quantities and qualities of leather can be manufactured. The methods and materials described herein can address many of the previously described problems with natural and engineered leathers, including those identified above.

Also described herein are engineered leathers that can address the problems by forming fiber-reinforced composite (FRC)-like materials, in which cells are cultured on fibrous supports (made up of fibers such as silk). ). In general, FRCs refer to composite building materials that form a class of high-performance materials used in various industries, including energy, construction, automotive, and sports. FRCs consist of a continuous matrix phase (usually a polymer matrix), a dispersed fiber phase (usually a stronger glass, carbon, or cellulosic fiber), and an interface between the matrix and the fibers. Within the interface, the fibers crosslink with the matrix phase to bind the material together and transmit forces from the matrix phase to the stronger fibers. As a result, materials with incredible strengths that are not possible with each material individually can be realized.

As mentioned, numerous tissue-engineered scaffolds have been developed over the last 25 years to construct biological tissues with defined structures and dimensions. These supports provide a surface area for cells to attach and grow tissue in three dimensions. Fibrous materials consisting of intertwined or woven fibers, 100nm to 100um in diameter, have been widely explored as tissue-engineered supports due to their large surface areas for cell growth per unit volume and high porosities to allow cell growth. the Cellular infiltration throughout the architecture of the 3D support. Tissue-engineered supports are typically biodegradable, allowing the tissue to replace the support as it grows. Therefore, the final product consists only of biological tissue to improve biocompatibility after implantation.

Tissue-engineered constructs are generally grown for biomedical applications, including insertion into a body to repair and/or replace biological tissue, so biocompatibility has been an important consideration. However, the use of biological tissues for consumer applications requires a very different set of considerations. In such cases, durability, appearance, and ability to be tanned or preserved should be considered. Described herein are methods and techniques for the fabrication of biological tissue, as well as the resulting engineered material, that may address the concerns outlined above. In particular, composite materials are described herein in which the fabric is grown along a fibrous support and cross-linked with the support during a process analogous to tanning to create a novel class of high-performance composite materials. , as well as methods for forming such composite materials. These engineered leathers can reproduce many of the structures and properties of natural leathers, but can be processed much more easily.

Disclosure Summary

The present invention, as claimed in claim 1, relates to a fiber-reinforced biological tissue composite material, the material comprising: a fibrous support comprising a plurality of fibers, in which the fibers are surrounded by a matrix extracellular, and wherein the extracellular matrix and the plurality of fibers are crosslinked with each other. Preferred embodiments of the invention are claimed in the attached dependent claims.

This document also discloses the following:

Reinforced engineered skins and methods of making these skins are described herein. These hides can be tanned to form leather materials, which can be used anywhere natural leather is used, including the manufacture of clothing (clothes, shoes, etc.), furniture (eg upholstery), or the like ( belts, bracelets, etc.).

For example, a method of manufacturing a reinforced engineered skin may include: placing a first plurality of collagen-releasing cells on a first surface of a mesh, the mesh having a first surface and a second surface; culturing the first plurality of collagen-releasing cells to form a first layer of collagen covering the first surface; flipping the mesh and placing a second plurality of collagen-releasing cells on the second surface of the mesh; and culturing the second plurality of collagen-releasing cells to form a second collagen layer covering the second surface, wherein the mesh is embedded between the first and second collagen layers and the first and second collagen layers are connected to each other through the mesh.

In general, placing the cells may include placing (eg, seeding) individual cells, groups/clusters of cells, and/or layers/sheets of cells within a collagen sheet that the cells have secreted. The cells can be placed directly on the surface, including on the surface of the previously placed mesh or layer.

In any of the variations described herein, the mesh may be (releasably) attached to a frame. The scaffold can be manipulated to transfer, including flipping, the mesh and any cell/collagen layers thereon. For example, placing the first plurality of collagen-releasing cells may comprise placing the first plurality of collagen-releasing cells on the first surface of the mesh where the mesh is releasably attached to a frame. The framework may remain on the cells throughout the hide manufacturing process and may be removed before, or in some variations after, tanning.

Any of these methods may include turning the mesh one or more times during the formation of the material, to apply additional layers of cells (and thus collagen) to the growing material. For example, the method may include flipping the mesh so that the second surface is facing up and placing at least a first additional plurality of collagen-releasing cells on top of the second surface and culturing the at least one additional first plurality of collagen-releasing cells. collagen to form at least one additional first layer of collagen covering the second surface. Alternatively or additionally, the method may include flipping the mesh so that the first surface is facing up and placing at least a second additional plurality of collagen-releasing cells on top of the first surface and culturing the at least a second additional plurality of collagen-releasing cells. of collagen to form at least a second additional layer of collagen on the second surface.

Thus, for example, the method may include sequentially flipping the mesh and placing at least a plurality additional collagen-releasing cells on top of the first or second surface and culturing the at least one additional second plurality of collagen-releasing cells to form at least one additional layer of collagen on the first or second surface.

As mentioned, the material can be tanned to form a leather. For example, the method may include tanning the material including the mesh embedded between the first and second layers of collagen. Tanning may include tanning the material having the mesh embedded between the first and second layers of collagen with the mesh removably held by a frame, where the mesh was secured to the frame during placement steps (for example, the step of placing the first plurality of collagen-releasing cells on the first surface of the mesh).

As mentioned, in some variations, placing the cells may include placing a sheet of cells (and collagen). A collagen sheet can be formed by growing the cells in a culture dish (or on another piece of mesh) and transferring the sheet to growing material including the mesh.

In some variations, the method includes placing the mesh on top of a preliminary layer of collagen-releasing cells in a culture dish so that the second surface of the mesh rests on the preliminary layer before placing the first plurality of collagen-releasing cells on top. the first surface of the mesh. This method can be particularly useful in variations where the mesh has relatively large pore sizes (eg, gaps through the mesh are greater than or equal to about 0.5 mm). Similarly, in some variations, the method may include placing a plug against the second surface of the mesh prior to placing the first plurality of collagen-releasing cells on the first surface of the mesh.

Any of the methods described herein may include the use of a mesh formed of a material to which cells do not normally adhere; despite not adhering, which can make it difficult for cells to grow and release collagen, cells can release collagen onto the mesh. This may be possible in some variations, due to relatively large pore sizes (eg larger than the diameter of the cells) so that cells adhere through mesh openings (eg to a substrate). , plug, or other layer of collagen/cells).

A method of manufacturing a reinforced engineered skin may include: placing a first plurality of collagen-releasing cells on a first surface of a mesh, the mesh having a first surface and a second surface; culturing the first plurality of collagen-releasing cells to form a first layer of collagen covering the entirety of the first surface; flipping the mesh and placing a second plurality of collagen-releasing cells on the second surface of the mesh; culturing the second plurality of collagen-releasing cells to form a second collagen layer covering the entirety of the second surface, wherein the mesh is embedded between the first and second collagen layers and the first and second collagen layers are connected each other through the mesh; and sequentially forming additional layers of collagen on the first, second, or first and second surfaces of the mesh to form a reinforced engineered skin having the mesh sandwiched between a plurality of layers of collagen-dense regions.

Sequentially forming additional layers of collagen may include forming a reinforced engineered skin having the striated pattern including a plurality of layers of collagen-dense regions and at least one mesh layer.

A method of making a reinforced engineered skin may include: placing a first plurality of collagen-releasing cells on a first surface of a mesh, the mesh having a first surface and a second surface, wherein the mesh is securely attached. releasable in a frame; culturing the first plurality of collagen-releasing cells to form a first layer of collagen covering the first surface; flipping the frame and mesh and placing a second plurality of collagen-releasing cells on the second surface of the mesh; culturing the second plurality of collagen-releasing cells to form a second collagen layer covering the second surface such that the mesh is embedded between the first and second collagen layers and the first and second collagen layers are connected to each other through of the mesh; placing at least one additional first plurality of collagen-releasing cells on top of the second surface and culturing the at least one additional first plurality of collagen-releasing cells to form at least one additional first layer of collagen covering the second surface; flipping the scaffold and mesh and placing at least a second additional plurality of collagen-releasing cells on top of the first surface and culturing the at least a second additional plurality of collagen-releasing cells to form at least a second additional layer of collagen on the second surface; and remove the mesh from the frame.

Also described herein are any of the reinforced engineered skins made by the methods described. For example, a reinforced engineered skin may include: a plane-lying body having a volume normal to the plane, the volume comprising a plurality of layers of plane-lying collagen-dense regions within the volume for form a layered pattern of collagen-dense regions; and a reinforcing mesh within the flat body and extending in the plane of the body, wherein a first of the plurality of layers of collagen-dense regions is adjacent to the mesh on a first side and follows a contour of the first side of the mesh and a second layer of the plurality of layers of collagen dense regions is adjacent to the mesh on a second side and follows a contour of the second side of the mesh. Hides can be tanned.

The skins may be substantially free of epithelial cells or undifferentiated keratinocytes. In particular, the hides may be free of any vascular tissue (eg, blood vessels or the like), or debris thereof, such as would be found in endogenous leather.

In general, the thickness of each collagen-dense region layer may be between about 10 µm and about 200 µm (eg, between about 20 µm and about 150 µm). The pore size of the mesh can be, for example, between about 2 µm and 5 mm.

In any of the reinforced engineered skins described herein, the skin may include one or more colorants or pigments. In addition, the skin may be patterned, for example, including having an embossed pattern; in some variations, the pattern is the mesh pattern. In general, the mesh may comprise a flexible net-like structure having a pore size of less than 5 mm. In some variations, the mesh comprises a synthetic polymeric material.

According to the invention as claimed, a fibrous support on which cells are cultured is used to produce a fiber-reinforced biological tissue composite material as claimed.

In addition to the mesh reinforcement described herein, any of the disclosed methods and engineered textile materials (leathers) may include a fibrous matrix. The mesh itself may be a fibrous matrix or may be formed of a fibrous matrix. The mesh may be formed entirely (or, in some variations, partially) of a crosslinking material that can crosslink with the extracellular matrix that has been grown by the cultured cells during the tanning process, as described herein. For example, the mesh may be formed of a protein fiber comprising amine, carboxylic acid and sulfhydryl groups.

For example, the engineered reinforced leather materials (engineered leathers) described herein may also or alternatively include a material composed of a fibrous matrix that has been tanned to allow cross-linking of the fibrous matrix with the collagen formed. by cultured cells (eg fibroblasts). These engineered leathers may be referred to as fiber-reinforced biofabric composites. Methods of making such fiber-reinforced biological tissue composites are also described herein.

For example, one method of forming a fiber-reinforced biological tissue composite material may include forming the material by culturing tissue-producing cells (eg, fibroblasts) on a fibrous support having amine (-NH ² ) and carboxylic acid (-) functional groups. -COOH), which can be cross-linked (eg by tanning) with tissue and/or proteins, such as collagen, formed and/or secreted by cells and then tanning (eg chemical cross-linking). Tanning can be done once the fibers of the support have been at least partially covered with the cultured cells and the extracellular matrix released by the cultured cells. In general, the supports described herein are fibrous supports formed of any crosslinkable material, but particularly proteinaceous materials (eg, containing amine and carboxylic acid groups), such as silk. Silk is just one example of a fibrous support that can be crosslinked; silk is usually made up of a protein fiber which may be mainly made up of fibroin. For example, silk fibers from farmed silkworms typically have a triangular cross-section with rounded corners, 5-10 µm wide. The fibroin heavy chain is mainly composed of beta sheets, due to the repeating amino acid sequence of 59 monomers with some variations. Silkworm fibers are naturally extruded from two silkworm glands as a pair of primary filaments ( brin), which are glued together, with sericin proteins acting as glue, to form a silk thread. silk ( bave). The diameters of the silk thread to form tussah silk can reach 65 |im. The silk emitted by a silkworm may consist of two main proteins, sericin and fibroin, with fibroin being the structural center of the silk, and sericin being the sticky material that surrounds it. Fibroin is composed of the amino acids Gly-Ser-Gly-Ala-Gly-Ala and forms beta pleated sheets. Interchain hydrogen bonds form, and side chains form above and below the plane of the hydrogen bond network.

Generally, a mesh is a network of material (yarns, cords, strands, fibers, etc.) that are connected, for example, by weaving or otherwise. The mesh may have pores of regular or irregular size, and/or regular or irregular shape, and/or regular or irregular spacing, and/or regular or irregular pattern. Generally, the mesh can be two-dimensional (eg, forming a sheet or surface); meshes are also contemplated three-dimensional As an example, the pores may be between 10nm and 5cm in one or more of; diameter or spacing. For example, the pore size may be generally between a diameter less than about 10nm, 20nm, 50nm, 100nm, 200nm, 300nm, 400nm, 500nm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1, 8mm, 1.9mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 12mm, 15mm, 2cm, 2.5cm, 3 cm, 4 cm, 5 cm, etc., and an upper diameter of about 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 0.6 mm, 0.7 mm, 0, 8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm , 1.9mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 12mm, 15mm, 2cm, 2.5cm, 3cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, etc., where the bottom diameter is always smaller than the top diameter. The strands (eg, fibers, yarns, wefts, etc.) or the material that forms the mesh can have diameters between about 100nm and 5mm in diameter (or in some cases more). A mesh can act as a support; As used herein, the support can be, but need not be, a mesh.

As mentioned, in any of the methods and apparatus (eg, engineered leathers) described herein, a support can be a protein fiber containing amine and carboxylic acid groups, such as cellulose fibers that are naturally occur containing (or modified to contain) amine and carboxylic acid groups. The fibers that form the backing can be chemically modified to enhance crosslinking of the fabric with the backing. For example, the support can be chemically modified to contain tissue crosslinking groups, including amines, carboxylic acids, sulfates, aldehydes, hydrazides, sulfhydryls, epoxides, acrylates, etc. These tissue crosslinking groups can be protected during tissue growth and are activated for crosslinking when tissue growth is complete. In particular, the cross-linking referred to herein is tanning, which may be identical to, or derived from, traditional tanning methods and techniques, including omitting those steps made unnecessary by the use of the tissue culture as described herein. In addition, an additional reinforcing crosslinking step can be used to crosslink chemical compounds not involved in the traditional tanning process (any other than amine and carboxylic acid groups). Traditional tanning chemicals can then be used to give the fiber-reinforced biofabric composite material a leather-like aesthetic. The supports can also be made of (and/or can include) carbon fibers, which can also be modified as discussed above.

In general, these tissue crosslinking groups may be pendant to the support with a spacer between 10 daltons and 100 megadaltons. The support can be cross-linked with the tissue through non-covalent interactions including ionic, hydrophobic and van der Waals forces. Alternatively or additionally, the support may be cross-linked to the tissue through covalent bonds. For example, the support can be directly reacted with amine or carboxylic acid groups on the fabric. The support can be reacted with a crosslinking agent that reacts with amine or carboxylic acid groups on the fabric. The molecular weight of the crosslinking agent can be between 10 daltons and 100 megadaltons. Tissue referred to herein is cultured cells and/or products released by those cultured cells (eg, extracellular matrix proteins, particularly collagen). Any of the crosslinking agents described herein may include a crosslinking agent functionality of between 2 and 2000.

The support can be made of fibers. As mentioned, the fibers may be of an appropriate dimension or size (eg, the fibers may be between about 100 nm and 1 m in length). The fibers can be joined in a woven or nonwoven architecture (or a combination of both). The density of the fibers in the support can be between 10 and 10,000 mg/cc. The porosity of the fibrous support can be between 10 and 99%.

Any suitable cell can be cultured on the fibrous support. The cells may originate from a tissue and/or cell line. For example, the cells may be of mammalian (eg, bovine, porcine, ovine, etc.) origin. The cells may be of reptilian origin (eg, snake, lizard, etc.). The cells may be of avian origin (eg, chicken, ostrich, turkey, etc.). The cells may originate from a fish (eg, shark, etc.). The cells may be of amphibian (eg frog, salamander, etc.) origin. The cells may be genetically modified (eg, to increase ECM production, including, eg, collagen, etc.) or may be unmodified.

Brief description of the drawings

Figure 1 shows an overview of a method for manufacturing engineered leather. In this example, cells are taken from an animal through a simple biopsy and then isolated and multiplied in cell culture medium. The cells are then allowed (and/or induced) to produce collagen, just as they would naturally after the cells spread so that the collagen forms sheets. The sheets are then treated to prevent shrinkage, and the thin sheets that do not shrink (eg, decellularized) are stacked on top of each other after seeding with additional collagen-forming cells, to form thicker sheets that are allowed to spread. adhere through the action of collagen-releasing cells. Finally, leather is formed from this multilayer structure using an abbreviated tanning procedure to modify the collagen.

Figures 2A-2C illustrate an example of forming a stack of collagen sheets to create a reinforced engineered leather. In this example, a mesh material (any reinforcing material) may be added within the stack/between the layers and the stacks (and/or individual cells seeded on the layers) may be grown on the reinforcing material, thereby encapsulating way the reinforcing material inside the material. This technique can be particularly effective with large mesh size openings.

Figure 3A shows an example of a mesh material on a scaffold on which cells can be grown.

Figure 3B schematically depicts an exemplary method for making reinforced engineered leather, as described herein, in which cells are grown sequentially on a first side of a mesh and the mesh (with cells grown and collagen) can be inverted to grow cells on the opposite side.

Figure 4 is a schematic illustration of a variation of a method of growing reinforced leather.

Figure 5 is another schematic illustration of a method of growing reinforced leather on a mesh.

Figure 6 is another schematic illustration of a method of growing reinforced leather on a mesh.

Figure 7 is a schematic illustration of a method of growing reinforced leather on a mesh.

Figures 8A and 8B show sectional and top views, respectively, of an example of a low resolution section through an example of a reinforced leather made using methods such as the method schematically illustrated in Figure 4 or Figure 5 , previously.

Figure 8C shows the engineered leather of Figures 8A and 8B in which an outer layer of material has been partially peeled away to reveal the mesh beneath.

Figure 9 schematically illustrates a section through an example of the reinforced leather as described herein. The figure is not shown to scale.

Figure 10 shows a first view of an example of a fibrous support formed by silk fibers that can be used to form the composite leather described herein. Figure 10 shows the image at reduced magnification (scale bar is 1 mm).

Figure 11 shows the fibrous support of Figure 10 at higher magnification (scale bar is 0.5 mm). Figures 12A and 12B illustrate tissue ingrowth on a fibrous support such as the silk support shown in Figures 10A-10B. Figure 12A shows the fibrous support and Figure 12B shows the fibrous support of Figure 12A after four weeks of fibroblast culture. Cells were seeded on the silk fiber support shown in Figure 12A and after four weeks of culture, the fiber support is surrounded by tissue (Figure 12B).

Figure 13 shows an example of a silk support on which fibroblasts are cultured, before tanning. In Figure 13, a section of the support on which fibroblasts have been cultured (for example, for four weeks) has been stained with picrosirius red to visualize collagen.

Figure 14 is a scanning electron micrograph showing a portion of a fibrous silk scaffold on which fibroblasts have been grown and allowed (and in some variations stimulated) to secrete collagen. Collagen-rich tissue has been grown throughout the silk fiber support.

Figure 15 shows examples of four different composites of fibrous supports (silk, high density PLLA, polyester, low density PLLA) and fibroblasts after eight weeks of culture, followed by tanning. Only the fibrous silk backing composite was successfully tanned, and only the fibrous silk backing includes amine and carboxylic acid groups that can crosslink with the fabric during the tanning process.

Fig. 16 shows an example of the leather-like surface of a fibrous silk backing composite material after tanning.

Figure 17 is an example of the edge of a fibrous silk backing composite material after tanning, where a gradient of weave has occurred towards the edge of the silk backing, revealing the silk fibers dispersed throughout the weave matrix. .

Detailed description

In general, reinforced engineered biomaterials and methods for making them are described herein. For example, described herein are engineered skins (eg, leather) that incorporate a support material such as a mesh or support embedded within the skin, as well as methods for making these reinforced engineered biomaterials. In some variations, the engineered skins described herein can be formed by forming sequential layers of collagen-releasing cells (eg, allowing them to grow and release collagen to form one layer) and combining one or more layers of collagen-releasing cells. collagen so that they can fuse with a support material such as mesh. Alternatively or additionally, engineered hides can be formed by adding collagen-releasing cells to a fibrous support formed of a material that can be cross-linked during tanning directly with the released extracellular matrix (eg collagen). Thus, the scrim and/or backing can be treated or otherwise formed with a material that can be cross-linked with the ECM during tanning.

In some variations, the engineered skins described herein can be formed by sequentially seeding collagen-releasing cells or layers of collagen-releasing cells onto a support material, such as mesh, and allowing the cells (or layer of collagen-releasing cells) to form. cells) to grow and release collagen onto the support material. The cells or cell layers are applied in such a way as to allow them to receive nutrients; they can be allowed to mature to form the new layer (including a new layer of collagen) before forming a new layer on top of these cells.

In any of the methods described herein, the mesh may be provided as a support and, in forming the layer, the mesh material may be periodically turned over so that preformed cells or layers of cells may be placed on top of either side. first second, and allowed to grow and adhere (releasing collagen) before turning the material and growing skin over to apply to the opposite side of the material.

As mentioned in the background section above, tissue engineering technology offers new opportunities to produce animal hides and skins (and leather from them) that are not associated with the environmental degradation of animal husbandry. livestock. Tissue engineering procurement has been defined as an interdisciplinary field that applies engineering and life science principles toward the development of biological substitutes that restore, maintain, or improve tissue or whole organ function. Langer R, Vacanti JP, Tissue Engineering, Science 260(5110):920-926 (1993 May). Tissue-engineered products fabricated using traditional materials and methods are limited in size due to the short distances that gases and nutrients can diffuse to nourish interior cells, typically thicknesses of engineered constructs are less than about 250 mm. -300 |im. Furthermore, existing techniques do not provide adequate speed and performance for mass production of engineered products. As a result, existing tissue engineering methods result in unattractive thin sheets and pastes on a commercially unfeasible scale. Thus, one objective of the hide/hide, or animal leather, and the methods for making the same described herein is to provide commercially viable and attractive animal hide, hide, or leather. Another goal is to provide high-throughput methods that can be reliably, accurately, and reproducibly scaled to commercial levels. Advantages of animal hide, hide, or leather and the methods for manufacturing them described herein include, but are not limited to, reproducible, high-throughput, and easily scalable production of personalized fabrics while maintaining a precise control of pattern formation, particularly in cases of multiple cell types, which can result in engineered animal hide, hide, or leather with attractive appearance, texture, thickness, and durability.

Engineered animal hide and leather and methods for producing them are disclosed herein. In certain embodiments, engineered animal hides, pelts, or hides comprising one or a plurality of layers of animal cells comprising one or more types of skin cells are disclosed herein, wherein said animal cells are grown in vitro. In certain embodiments, each layer of animal cells provided herein is biofabricated. A structural support, such as mesh, can be incorporated into any of the engineered materials described herein.

Also described herein are engineered animal hides and skins and methods for producing them, including a fibrous support that can be used to grow cells that can release eCm that can cross-link the fibrous support during tanning. For example, the fibrous support can include available reactive groups that can crosslink with one or more ECM proteins (eg, collagen).

For example, the methods described herein may include first forming collagen sheets by growing (cultivating) collagen-releasing cells and allowing (and/or stimulating) them to secrete collagen. In culture, the cells can be grown to confluency on a substrate such as the bottom of a cell culture dish, a flask, a roller, a chamber (eg, roller chamber), or the like, and/or on a fibrous support.

In general, the collagen-releasing cells described herein may be derived from tissue extracts/explants, immortalized cell lines, or engineered (transgenic) cell lines, or any variations thereof. In some variations, the cell can be grown to complete confluency (eg, 100% confluency), where further growth of the cells is inhibited but they can continue to produce or can be stimulated to produce and release collagen. In some variations, cells may not be grown to complete confluency (eg, about 99% confluent, 95% confluent, 90% confluent, 85% confluent, 80% confluent, etc. ). Cells can be grown to greater than 80% confluency, greater than 85% confluency, greater than 90% confluency, greater than 95% confluency, and/or just below full confluency (100%).

As described in greater detail below, and in particular with reference to Figures 3-8C, cells and cell sheets can be grown directly on a support material (eg, mesh); Alternatively, cells may be initially grown on one or more sheets that are combined with a support material (for example, mesh) and then allowed to grow further (for example, by adding additional cells or sheets so that release collagen).

In general, growing structures (eg, sheets) that include collagen can be treated to prevent contractions. Cultured cells, including collagen-releasing cells, may begin to contract during culture. The cultured cells that form the multiple collagen sheets can be treated during growth and/or towards the end of growth. Normally, the collagen sheets are treated before contraction begins or before a contraction greater than a threshold occurs. In general, it is desirable to avoid shrinkage because shrinking cells in a collagen sheet can cause the sheet to deform, which can be undesirable when forming an engineered leather. In some variations, treatment of the collagen sheets or treatment of the cells that form the collagen sheets may include destruction of the cells. Therefore, the collagen sheets including the cultured cells can be treated by decellularizing (eg, removing or destroying) the collagen-releasing cells. In some variations, this may include treating the collagen sheets with ethanol. After treatment, the resulting collagen sheets may be referred to as "non-contractile" sheets or "decellularized" sheets (for sheets treated to destroy and/or remove collagen-releasing cells).

After treating the sheets to form non-contractile sheets, the sheets may be washed (eg, rinsed or the like) to remove material used to treat the cells. Other additional treatments may also be performed. After that, an engineered leather of a desired thickness can be formed using the non-shrink sheets. For example, the sheets can be stacked sequentially or simultaneously and adhered to each other. Adhesion can be achieved by re-seeding the sheets (eg, one sheet surface) so that collagen-releasing cells can communicate with one or both sides of the non-contractile sheets. This second group of cells may be the same cells used to grow the collagen sheets, or they may be different cell types (although also collagen-releasing) or different cell type distributions in which multiple cell types are used to grow collagen sheets. A support material, such as mesh, can be applied between sheets or over a single sheet on which additional collagen-releasing cells are grown by themselves or under another sheet.

By adding each new layer of cells or mesh and collagen-releasing cells (or layer of cells and released collagen), then the second group of collagen-releasing cells can be allowed to grow (eg cultured) until adhesion is obtained. enough between the two sheets. Sufficient adhesion can be determined by time in culture (for example, approximately: 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.), or it can be determined empirically. For example, the "stack" of sheets (two sheets that are adhered to each other by the second seeded group of collagen-releasing cells) may be allowed to grow for more than about 4 days, more than about 5 days, more than about 6 days, more than about 7 days, more than about 8 days; and/or less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, etc., expressly including between about 2 and 9 days, between about 4 and 7 days, about 5 days, etc.

In some variations, a filler material may be inserted between the sheets before they are grown to allow them to adhere. Any suitable filler material can be used, but particularly collagenous filler material. The filler material may be useful for increasing the thickness of the stack that forms engineered leather and/or reducing the number of collagen layers used to form engineered leather. engineering. Examples of filler materials include reconstituted collagen and/or collagen pulp (eg mechanically cut collagen). Reconstituted collagen can be depolymerized (eg collagen monomers or small polymers). Filler material can be added to a desired thickness. In some variations, the filler material may be seeded with cells from the second group of collagen-releasing cells, in addition to or alternatively to the seeded collagen-releasing cells on one or both layers that are to be stacked together around the filler material.

When additional collagen laminae are added to a stack (eg, non-contractile collagen laminae) forming an artificial leather, additional laminae may be added sequentially or in parallel, or a combination of both, where a small number (eg, fewer (about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, 2, etc.) of sheets can be stacked on top of a first non-contractile sheet of collagen and can be allowing the sheets to adhere by culturing the second set of collagen-releasing cells placed between the two or more sheets (with or without filler material) with or without the reinforcing material (eg, mesh). In this way, a "stack" of collagen sheets that have adhered can be formed. After that, the dimensions (e.g. thickness) of the pile can be increased by combining two or more piles and allowing them to adhere by growing one (or more) on top of the other with or without fill material after seeding them with part of the second group of collagen-releasing cells.

cells

Many types of cells can be used for the collagen-releasing cells used to produce the collagen sheets/layers and thus the engineered hide, hide and leather products described herein. The collagen-releasing cells may be homogeneous (eg, cell type) or may be a mixture of collagen-releasing cells and/or cells that release other ECM materials and/or cells that do not release collagen. In some embodiments, engineered animal hide, hide, and leather products are designed to resemble traditional animal hide, hide, and leather products and the cell types are chosen to approximate those found in engineered animal hide, hide, and leather products. skin, hide and leather of traditional animals. In further embodiments, the engineered animal hide, hide, and leather products, and sheets/layers, include animal epidermis, basement membrane, dermis, hypodermis, scale, shield, osteoderm, or a combination thereof. In some embodiments, the animal epidermis provided herein comprises stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum germinativum, stratum basalis, or a combination thereof. In some embodiments, the animal dermis provided herein comprises stratum papillary, stratum reticularis, or a combination thereof. In some embodiments, the animal scales provided herein comprise placoid scales, cosmoid scales, ganoid scales, elasmoid scales, cycloid scales, ctenoid scales, crenate scales, spinoid scales, or a combination thereof.

In certain embodiments, the animal cells provided herein comprise epithelial cells, fibroblasts, keratinocytes, comeocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. In some embodiments, the epithelial cells provided herein comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. In some embodiments, the fibroblasts provided herein are dermal fibroblasts. In some embodiments, the keratinocytes provided herein are epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof.

In certain embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is from about 20:1 to about 3:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is from about 20:1 to about 4:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is from about 20:1 to about 5:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is from about 20:1 to about 10:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is from about 20:1 to about 15:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 25:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 24:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 23:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 22:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 21:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 20:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein document is approximately 19:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 18:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 17:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 16:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 15:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 14:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 13:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 12:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 11:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 10:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 9:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 8:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 7:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 6:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 5:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 4:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 3:1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 2:1.

In certain embodiments, the animal cells provided herein are substantially free of epithelial cells, fibroblasts, or undifferentiated keratinocytes.

In other embodiments, the engineered animal skin, hide, or leather products include neural cells, connective tissue (including bone, cartilage, cells that differentiate into bone-forming cells and chondrocytes, and lymphatic tissues), epithelial cells (including endothelial cells that form linings in cavities and vessels or channels, exocrine secretory epithelial cells, absorptive epithelial cells, keratinizing epithelial cells, and extracellular matrix secreting cells), and undifferentiated cells (such as embryonic cells, stem cells, and other precursor cells), among others.

In certain embodiments, the engineered animal skin, hide, or hide further comprises an extracellular matrix or connective tissue. In certain embodiments, the engineered animal skin, hide, or hide further comprises one or more components selected from the group consisting of collagen, keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin , dermatopontin, lipid, fatty acid, carbohydrate, and a combination thereof.

In some embodiments, the cells are obtained from commercial sources. In certain embodiments, the cells are derived, by way of non-limiting examples, from mammals, birds, reptiles, fish, crustaceans, molluscs, cephalopods, insects, non-arthropod invertebrates, and combinations thereof. In some embodiments, the animal cells are human cells. In certain embodiments, the animal cells provided herein are non-human cells. In some embodiments, suitable cells are derived from mammals such as antelope, bear, beaver, bison, warthog, camel, caribou, cat, cow, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, goat wild, kangaroo, lion, llama, lynx, mink, reindeer, ox, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, and zebra, or combinations thereof. In some embodiments, suitable cells are derived from birds such as chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, and turkey, or combinations thereof.

In some embodiments, suitable cells are derived from reptiles such as turtle, snake, crocodile, and alligator, or combinations thereof.

In some embodiments, suitable cells are derived from fish such as anchovy, sea bass, catfish, carp, cod, eel, flounder, puffer fish (fugu), grouper, pollock, halibut, herring, mackerel, dolphinfish, stingray, marlin, roughy orange perch, pike, salmon, sardine, shark, snapper, flounder, skate, swordfish, tilapia, trout, tuna, and walleye, or combinations thereof.

In some embodiments, suitable cells are derived from amphibians such as frog, toad, salamander, newt, or combinations thereof.

In some embodiments, suitable cells are derived from crustaceans such as crab, lobster, prawn, and shrimp, or combinations thereof.

In some embodiments, suitable cells are derived from molluscs such as abalone, clam, conch, mussel, oyster, scallop, and snail, or combinations thereof.

In some embodiments, suitable cells are derived from cephalopods such as cuttlefish, octopus, and squid, or combinations thereof.

In some embodiments, suitable cells are derived from insects such as ants, bees, beetles, butterflies, cockroaches, crickets, damselflies, dragonflies, earwigs, fleas, flies, grasshoppers, mantises, mayflies, moths, silverfish, termites. , wasps or combinations thereof.

In some embodiments, suitable cells are derived from non-arthropod invertebrates (eg, worms) such as flatworms, tapeworms, trematodes, roundworms, roundworms, hookworms, segmented worms (eg, earthworms, polychaetes, etc.), or combinations thereof.

In some embodiments, engineered materials (eg, animal hide, hide, or leather products) include one or more additives in addition to the support material(s). For example, one or more additives are selected from: minerals, fiber, fatty acids, and amino acids. In some embodiments, in engineered animal hide, hide, or leather products, the sheets/layers include one or more additives to enhance commercial appeal (eg, appearance, color, odor, etc.). In further embodiments, in engineered hide, hide, and leather products, the sheets/layers include one or more colorants and/or one or more odorants.

In some embodiments, in engineered animal hide, hide, or leather products, the engineered sheets/layers include one or more of: matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. The term "growth factor" as used herein refers to a protein, polypeptide, or complex of polypeptides, including cytokines, that is produced by a cell and that can affect itself and/or or to a variety of other neighboring or distant cells. Normally, growth factors affect the growth and/or differentiation of specific cell types, either during development or in response to a multitude of physiological or environmental stimuli. Some, but not all, growth factors are hormones. Exemplary growth factors are insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), growth factors of fibroblasts (FGF), including basic FGF (bFGF), platelet-derived growth factors (PDGF), including PDGF-AA and PDGF-AB, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a) , transforming growth factor beta (TGF-P), including TGFpi and TGFP3, epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF) , interleukin-6 (IL-6), IL-8, and the like.

In some embodiments, in engineered animal hide, hide, or leather products, the engineered sheets/layers include one or more preservatives known in the art. In some embodiments, the preservatives are antimicrobial preservatives including, by way of non-limiting examples, calcium propionate, sodium nitrate, sodium nitrite, sulfites (eg, sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc. ) and disodium ethylenediaminetetraacetic acid (EDTA). In some embodiments, the preservatives are antioxidant preservatives including, by way of non-limiting examples, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

Skin, hide and leather of animals obtained through engineering

Engineered animal skin, hide or leather products (eg, made using these engineered hides) are disclosed herein. In some embodiments, the engineered animal hide, hide, or leather products are further processed by any method known in the art. Examples of known processing methods include processing by preserving, soaking, liming, dehairing, fleshing, splitting, deliming, reliming, rendering, degreasing, pilling, bleaching, pickling, acid and salt removal, tanning, thinning, retanning , lubrication, semi-finished leather forming, wetting, watering, shaving, rechroming, neutralizing, dyeing, oiling, filling, peeling, stuffing, bleaching, fixing, hardening, drying, conditioning, grinding, stacking, burnishing, finishing, oiling, brushing, padding, impregnation, spraying, roller coating, curtain coating, polishing, plating, engraving, ironing, enameling and tumbling. It is important that the methods described herein do not require any of the pre-processing steps that are necessary when using natural animal hide, including unhairing (dehaired), liming, fleshing, splitting, deliming, reliming, etc. Layered bodies formed as described herein may be formed of any appropriate length, and collagen (and other ECM molecules) may be formed by the cultured cells, resulting in a layered body that does not include structures such such as hair follicles, blood vessels, muscle (eg arrector pili muscle), etc.

In general, the engineered leather described herein may be tanned (or processed by a similar process) to modify the extracellular matrix material. As discussed above, one of the main components of the ECM is collagen (particularly type I collagen). Tanning can modify collagen. For example, one tanning agent, chromium(III) sulfate ([Cr(H2O)6]2(SO4)a), has long been considered the most efficient and effective tanning agent. Chromium(III) sulfate dissolves to give the hexaaquachrome(III) cation, [Cr(H ² O) ⁶ ]3+, which at higher pH undergoes processes called olation to give polychrome(III) compounds that are active in tanning, being the crosslinking of collagen subunits. Some ligands include the sulfate anion, the carboxyl groups of collagen, the amine groups of amino acid side chains, as well as masking agents. Masking agents are carboxylic acids, such as acetic acid, which are used to suppress polychrome (III) chain formation. Masking agents allow the tanner to further increase the pH to increase collagen reactivity without inhibiting the penetration of chromium(III) complexes. The high hydroxyproline content of collagen allows for significant cross-linking via hydrogen bonds within the helical structure. Ionized carboxyl groups (RCO ² -) are formed by hydrolysis of collagen through the action of hydroxide. This conversion can occur during the lime process, before the introduction of the tanning agent (chrome salts). The ionized carboxyl groups can coordinate as ligands with the chromium(III) centers of the oxohydroxide groupings. Tanning can increase the separation between protein chains in collagen (eg, from 10 to 17 A), consistent with crosslinking by polychrome species, of the type arising from olation and oxolation. Chromium can crosslink with collagen. Chromium's ability to form such stable bridges explains why it is considered one of the most efficient tanning compounds. Chrome tanned leather may contain between 4 and 5% chrome. This efficiency is characterized by its greater hydrothermal stability of the leather and its resistance to shrinkage in hot water. Other tanning agents can be used to tan the layered body and modify the collagen.

In some embodiments, the engineered animal skin, hide, or leather products are substantially free of pathogenic microorganisms. In additional embodiments, controlled and substantially sterile methods of cell preparation, cell culture, sheet/layer preparation, and preparation of engineered animal skin, hide, or hide result in a product substantially free of pathogenic microorganisms. In further embodiments, a further advantage of such a product is increased utility and safety.

In some embodiments, the engineered animal skin, hide, or leather products are shaped. In further embodiments, the animal skin, hide, or hide is formed, for example, by controlling the number, size, and arrangement of the non-shrinkable sheets/layers used to make the animal skin, hide, or hide. In other embodiments, animal skin, hide, or hide is formed, for example, by cutting, pressing, molding, or stamping. In some embodiments, the shape of the animal skin, hide or leather product is selected to resemble a traditional animal skin, hide or leather product.

support materials

Any of the engineered materials described herein may include a support material. Support materials can include any non-biological exogenous material. Although other materials may be used, meshes, including mesh sheets that are woven, extruded, or the like, and that include openings (eg, pores) therethrough, are of particular interest. In general, the embedding of support materials can modify the physical properties of the leather and can also be done in a way that enhances the ability to make larger sizes (eg surface areas) of the material.

For example, an exogenous, non-biological, textured component may be incorporated or embedded in the engineered leather. The support material (or component) may act as a support for the living cells (or a layer of living cells), and the cells/cell layer may adhere to the support and secrete extracellular matrix, thus embedding it completely.

Examples of support materials include materials of animal origin (hair, silk, etc.), materials of plant origin (cotton, fine yarn, sisal, hemp, etc.), materials derived from polymers, petroleum/plastics (for example, polystyrene, polyester, Kevlar, polyethylene, polypropylene, polyurethane, nylon, etc.) and other polymeric materials (eg poly(lactic acid), polyhydroxyalkanoates, etc.), metals, minerals (jewels, rocks, etc.), allotropes carbon (for example, graphene, nanotubes, fullerenes, etc.), or the like, including combinations of these.

In some variations, the geometry of the support material may be a substantially two-dimensional layer of material (eg, having a length and width much greater than the thickness), such as a 2D mesh. The mesh can be created by any suitable method, including weaving (for example, knitting, felting, braiding, lacing, etc.), extrusion, casting, cutting, or the like. For example, weaving may include weaving with fibers

perpendicular to each other or at a variety of angles. Other examples of 2d ^meshes include those created using

various methods of engraving, drilling, printing, expanding, etc. Meshes can be created by

3D printing of flat, curved or patterned meshes.

The 2D mesh can be modified to fit a 3D curved surface (3D composite material), or it can be

substantially flat. The mesh size and area coverage (including, for example, the area of holes or

strands to total area) can range from micrometers to millimeters. For example, the size of the

openings in the mesh can vary from 0 (close tissue) to just <100% (very open tissue). In addition, the

thickness of the 2D mesh can vary, for example, from atomic dimensions to millimeters. In general, the

openings through the mesh can be called the pore size of the mesh. The pore size of the mesh

("pore size") can be uniform or variable. As described in more detail below, different

Mesh pore sizes may interact differently with cell cultures as shown.

described in this document.

Although other support structures can be used instead of, or in addition to, meshes, there are numerous

advantages when using a support structure comprising a mesh. When the textile material obtained

by engineering (for example, leather), collagen-releasing cells can attach to both sides of the

mesh, and the entire mesh can be easily encapsulated, while remaining flexible and enhancing durability and other

properties. This adhesion can also help in the fabrication of the mesh. For example, cells can

adhere to mesh surfaces. Cells can be coated on both sides of the mesh,

wrapping the mesh. As illustrated below (for example, Figures 8A-8C), the cells that make up the

engineered hide may be connected by small fragments that grow into the pores or

spread between the pores. Although non-mesh (and non-porous) materials can be used, which allow the

cells grow on both sides as described herein, it is beneficial to use a mesh

having pores that allow cell/layer fusion on either side of the mesh. Therefore, even the

meshes that are made up of relatively non-adherent materials, to which the cells themselves do not adhere.

adhere easily, they can be used as a support, as the cells/layers of cells used to form the skin

engineered material can be bonded through the pores. In general, the material that forms the structure of

support may be biocompatible, however, it is not required to provide a surface on which to

cells adhere (or requires an intermediate adherent surface). Therefore, in general, the mesh can

be incorporated or encapsulated in (surround) the engineered hide and/or leather. So the methods

described herein allow for a broader range of materials that can be used to form the

composite engineered hide/leather than those previously examined.

For example, in some variations, the mesh support material (or the "mesh") may comprise a structure

The mesh can be elastic or quite rigid. For example, the mesh can have an elasticity that varies from

stiff up to various values of elongation (allowing stretching), and bending stiffness can vary

from rigid to flexible. In general, the mesh can withstand at least one method of sterilization before

be added to the sterile cell culture environment (e.g. heat sterilization, steam sterilization,

radiation sterilization, etc.). In addition, the support structure (eg mesh) can be chemically

inert and can withstand chemical treatment(s) without loss of structural integrity or material properties,

including resistance to the tanning process. For example, the mesh can withstand a wide range of pH

in aqueous solution.

Any of the mesh materials described herein can promote cell attachment, or

by its inherent surface properties or be achieved by surface preparation (e.g.,

coating, functionalization, etc.). Alternatively or additionally, as mentioned above, the material

mesh can be trapped in the leather structure during assembly without requiring cell attachment to the

mesh material itself.

Examples of methods of incorporating a mesh material are described below with reference to the figures.

within hides and/or hides formed from the engineered hides. For example,

Figures 1 and 2 schematically illustrate a method of forming an engineered leather that includes the

use of a mesh to form a final engineered skin that is reinforced with the mesh material.

Figures 3-7 illustrate three alternative methods in which cells are grown on the mesh and layers of

collagen (released by the layers of cells formed) are formed directly on the mesh, which can also

act as a support during the manufacturing process.

In Figure 1, a tissue explant (eg, taken from an animal such as a cow) (see, eg, US-2013-0255003-A1 or PCT Application No. PCT/US2014/042384, filed June 13, 2014). The cells (skin cells) can be removed through a simple biopsy and then isolated and multiplied in cell culture medium. In particular, cells can be cultured in a medium such as a layer and allowed or induced to secrete collagen; the cells can be spread out so that the collagen forms sheets, as illustrated. The sheets can then be treated to prevent shrinkage, and the non-shrinking (eg, decellularized) thin sheets can be stacked on top of each other after seeding with additional collagen-forming cells. As part of this procedure, the mesh layer can be added between the layers and also seeded with cells. This procedure can result in the formation of thicker sheets of engineered material. In one example, a mesh is placed over a preformed sheet of collagen and cells are seeded onto it and allowed to grow and secrete collagen; the cells and secreted collagen, while primarily organizing in the upper layer (and at least partially incorporating the mesh), can also adhere to either or both of the mesh and the layer below the mesh. After adequate time for collagen to be secreted, additional layers may form on the upper and/or lower sides of the mesh. For example, one or more additional layers may be formed on top of the new layer by seeding new cells and allowing them to grow and release collagen, and/or additional preformed layers of collagen may be added on top of the growing pile (including the mesh) and the growth is allowed. merge the new layer. Additional cells may be added or the cells in the previous layer may act to anchor to the existing collagen layer(s) in the newly added layer. Periodically, the stack (which includes the mesh) can be flipped so that new layers can be added to the bottom (now top) of the stack. These procedures can be repeated as necessary to grow the stack to a desired thickness. Finally, leather is formed from this multilayer structure through an abbreviated tanning procedure to modify the collagen.

Figure 2 schematically illustrates a little more detail of a variation of the steps described above. In this example, thin layers of collagen are formed by growing them into sheets, then the sheets are combined with a mesh material between them and allowed to fuse. As before, shrinkage of cells when cultured on slides can be prevented by treating the cells (for example, killing them) or by other treatments, in which case it may be beneficial to add new cells to the slides to enhance cell fusion. collagen layers, as shown in Figure 2B. Although multiple layers can be added simultaneously in theory, in practice, diffusion can limit the growth capacity of cells when they are stacked thicker than two (or three) layers. Figure 2C illustrates an example of the flat layer of collagen released by the cell layers that can be fused together as described.

Alternatively or additionally, in some variations, the cells may be grown directly on and/or around a mesh material to be incorporated into the resulting textile material. For example, Figure 3A illustrates an example of a scaffold-supported mesh material (shown as round, although any shape may be used) that can be used to grow cells to form any of the materials described herein. In this example, the shell can be removed from the mesh, as the mesh will form an integral part of the resulting textile material (hide/leather). The scaffold and mesh can float or be submerged in a culture medium during tissue growth.

Figure 3B is a schematic illustration of a general method for forming engineered leather materials as described herein. In this example, cells may first be applied (eg, seeded) onto the first side of mesh 303. The mesh may be laid flat and submerged in culture medium. In some variations, the mesh is held in a frame as shown in Figure 3A. The mesh may be unsupported (accepts any scaffold) or may be backed by a supportive backing, such as a surface on which cells can grow and then be removed (e.g. agar, cell culture dish bottom, etc. ). Cells can then be cultured on the first (eg, top) side of the 305 mesh and allowed to grow and release collagen (eg, in some variation, grow to confluency or near confluency, as described above). ). For example, cells can be cultured for hours, days, or weeks (eg, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, etc.). 12 days, etc). Optionally, thereafter, additional cells (and/or a layer of collagen-releasing cells as described above) may be placed on top of mesh 307 and allowed to release collagen and further adhere to the growing pile. This stage can be repeated sequentially to expand the size of the stack. After that, the mesh can be turned 309, and the procedure is repeated on the second (now top) side. Thus, additional cells (and/or collagen layers or cells and collagen) can be placed on the second side 313 and cultured and allowed to release additional collagen for adhesion 315. As before, the cells and/or collagen layers (or additional collagen and cells) can be placed on this second side and cultured to expand the thickness of the material 317. The material can be flipped again and the steps repeated to continue growth of the material. Once the desired thickness is reached, the procedure can be stopped, and the material, now with the mesh embedded, can be removed and treated (eg tanned).

Alternatively, in some variations, multiple layers/stacks with embedded mesh can also be stacked on top of each other to form thicker layers. For example, a mesh can be grown with one or more layers as described in Figure 3B, and an additional layer of mesh can also be stacked on top of the growing material. growth and join it by adding additional cells (which then secrete collagen) onto the growing pile. Therefore, multiple mesh layers can be incorporated into a single material, enhancing the properties of the material.

Figure 4 illustrates a variation of a method as described above. In Figure 4, a relatively small mesh size is shown (for example, a pore size between 2 and 300 µm). Small mesh sizes can trap cells or cell aggregates (eg, typical cell size 10-20 microns, typical aggregate size 100-500 microns). As mentioned above, the mesh can be mounted on a frame (for example, a free-form, circular, rectangular 2D frame) that immobilizes and straightens the mesh, creating a smooth, flat, horizontal surface, as illustrated in Fig. figure 3A. The scaffolded mesh can be transferred to a container (histoculture Petri dish) containing histoculture medium, then seeded with cells as shown in Figure 4a. Cells can then adhere, spread, and secrete extracellular matrix (collagen) onto the upper side of the mesh (FIG. 4b). After a predetermined period of time (in which the cells release collagen to form a collagen layer), the scaffold can be inverted and the other side seeded with cells, as shown in Figure 4c. In figures 4b and 4c, collagen can also extend between the pores (not shown). The new cells can also adhere and secrete a layer of collagen on the other side of the mesh (figure 4d). This procedure can be repeated (Figures 4e and 4f) to achieve the desired thickness of the engineered material. The resulting material, with the mesh embedded, can be subsequently tanned. Note that this figure is not shown to scale.

In this example, small mesh sizes may allow cells (or groups of cells) to sit directly on the mesh, and collagen is still released between the pores (not shown), fusing the layers over both mesh sizes. and locking the position of the mesh. Therefore, they can be encapsulated even with materials to which cells do not normally adhere as shown.

For meshes with a slightly larger pore size (for example, meshes that have a pore size greater than 300 microns), cells can also be grown directly on the mesh, as shown in Figure 5. In Figure 5a , the cells are plated on a histoculture plate containing culture medium and cultured for a predetermined period of time, during which the cells secrete a layer of extracellular matrix (particularly collagen) as shown schematically in Fig. 5b. The unretained mesh is then placed on top of this cellular-extracellular-matrix layer and again seeded with cells (FIG. 5c). The number of cells in the second seeding can vary depending on the thickness and coverage area of the mesh. During the second incubation time interval (which can be the same as the first), cells from the second seed will divide and eventually fill the holes in the mesh while secreting extracellular matrix and coupling the mesh and cell/matrix layer. underlying extracellular as shown in Figure 5d. The seeding procedure can be repeated again (Figure 5e) to completely cover the mesh, creating a top layer of ECM, cultured for the same duration as the previous two layers (Figure 5f). The ECM sheet-mesh composite can then be separated from the plate and grown in a non-adhesive environment until ready for tanning, or additional layers can be made to increase thickness. For example, the mesh/ECM composite can be retained in the frame and further seeded by regularly reversing the frames to achieve an equal thickness coating on both sides.

Another example (also not shown to scale) is illustrated in Figure 6. In this example, a large pore size (eg greater than 500 |im (eg between 0.5mm and 5mm)) is used. . In Figure 6, the mesh is placed on a culture plate or substrate 601 (potentially including, as mentioned above, on top of an existing layer of collagen, eg, a decellularized layer). In this example, the cells are first grown directly on the substrate (FIG. 6a) and allowed to release collagen (FIG. 6b). A mesh (large pore size) is then applied as shown in Figure 6c. Cells (and/or an additional layer) are then applied over and onto the mesh, as shown in Figures 6c and 6d, and the cells are allowed to grow and release collagen. After that, additional layers can be added by sequentially adding cells and culturing them for growth, as illustrated in Figures 6e-6h. After that, the stack including the mesh can be removed from the plate (separating everything from the plate, as the cells adhere to the mesh better than the surface of the plate), and the mesh is turned over, and cells are applied. /additional layers, as shown in figure 6i.

Another variation using a large pore size mesh is shown in Figure 7. When the interaction between the cells and the culture plate may be stronger than the interaction between the cells and the mesh, separation of the composite structure from the culture plate (Figure 6f) may cause the cell/ECM layer to collapse. detach from the mesh, which is undesirable. Thus, in some variations, the mesh may be retained in a frame and a non-adhesive plug 703 (eg, Teflon, agarose, etc.) may be placed under and in contact with the mesh. The mesh can then be seeded with cells (FIG. 7a), where cells will accumulate in the openings and on the top surface of the mesh, secreting extracellular matrix (FIG. 7b). The plug can then be removed, and the mesh can be inverted and cells seeded on its other side (FIG. 7c). Sequential inversion, seeding, and cell culture can then be performed to deposit multiple layers on both sides to thicken the construct (Figures 7c, 7d, 7e, 7f).

Any of the composite mesh/ECM constructions described herein can be tanned either before or after release from the scaffold, or tanned while retained. Tanning while retained by the frame may be desirable where the tanning agents are excessively astringent and would distort the construction. Therefore, a releasable frame can be used, and be released after tanning. A proof-of-concept experiment is shown in Figures 8A-8C. In this example, a polyester-leather composite material was prepared by the method illustrated in Figure 6 above. After separating the structure from the culture plate, the composite material was further cultured for 3 weeks in a non-adhesive environment and subsequently tanned with conventional chrome tanning methods. Even after tanning, the collagen material completely covers the mesh with a thin layer of engineered leather, as shown in Figures 8A and 8B. 801 polyester mesh is encapsulated with tanned 805 collagen. A cross section shows uniform coverage over the wires 801 (FIG. 8A), completely filling the mesh (FIG. 8B). In Figure 8C, the resulting material was deconstructed by partially removing the top layer 813 prior to tanning (eg, by removing it from the plate) to illustrate the triple layer structure. The underlying mesh 815 is exposed to the right of the torn region 817.

Cells in emerging sheets formed as described above can secrete and assemble extracellular matrix. For each seeding, the rate of secretion and assembly may depend on the cell type and slows down once a certain thickness of the lamina is reached. This may be because either (i) the cells have exhausted their ability to produce extracellular matrix, (ii) the extracellular matrix layer prevents nutrients from the culture medium from reaching the cells, or (iii) the matrix is degraded. or remodeled by the enzymatic activity of the cell.

In response, the sheets being formed (eg, the layer of cells and extracellular matrix) may be seeded with freshly prepared collagen-releasing cells of either the same or a different type as the layer already formed. Normally, these new cells adhere tightly to the collagen in the underlying layer and begin to divide and produce new extracellular matrix. The above reseeding procedure can be iterated (ie, repeated) until the desired thickness of the emerging multilayer tissue construct is achieved.

In general, during the culture time, both the newly added cells and the initial layer cells can secrete collagen, resulting in the interconnection between the collagen layers. Secretion by cells in the initial layer may occur at a slower rate than by newly added cells as they move away from the culture medium on both sides of the mesh.

In some variations, two reseeds are performed leading to a three-layer structure on both sides of the mesh. In other variations, the number of reseeds may be greater than 3. Note that as the number of reseeds increases, the cells in the lower layers progressively move away from the interface of the construct with the nutrient-containing culture medium. This can lead to the slowing down of their cellular functions, particularly the release of collagen, and can eventually lead to their death by starvation.

In some variations, samples are additionally coated with a polymer to impart further functionality to the material, eg waterproofing, very high strength, chemical resistance, etc.

In general, each layer is formed by culturing the cells until a layer of collagen forms within the new layer. This may take, for example, about a week (eg about 5-10 days). For example, if a layer needs 10 days to grow, in some variations each side can be reseeded before the layer is fully grown, and the structure can be flipped over after the cells are sufficiently attached (within a few days). few days).

Any of the engineered materials (textiles, hides, leathers, etc.) described herein may have a distinct ultrastructure that includes the embedded material (eg, mesh), but is also layered, as a result of the method. manufacturing described. For example, Figure 9 schematically illustrates an ultrastructure of an engineered material having multiple layers in which a mesh material is embedded. In Figure 9, mesh 901 is embedded within a plurality of layers formed by any of the methods described herein. The result is layers in which a cross section shows layers of collagen dense regions 904 alternating with less dense regions 906 . The less dense regions may correspond to the cellular region and the adhesion region between the sequentially applied layers. This ultrastructure is different from native materials, both in the inclusion of the 901 mesh material and in the layering of the collagen.

The methods described herein represent a very flexible method of embedding virtually any support material (eg, mesh) into an engineered leather, which can substantially change the physical properties of the leather. Although the examples described herein are Specific to meshes, it should be understood that non-mesh materials (eg, yarns, etc.) may also be used.

fibrous supports

Also described herein are engineered leathers formed using a backing comprising a fibrous backing that is tanned (eg, cross-linked) with the cultured cells and/or any extracellular matrix (ECM) components released by the cells. cultivated that are formed. The resulting engineered leather may be referred to as a fiber-reinforced fabric composite material and may have superior properties (eg, durability, strength, etc.) compared to other engineered leathers. Also disclosed are methods of forming these fiber-reinforced fabric composites, including methods of growing/cultivating them and methods of tanning them.

In general, the supports described herein are configured to crosslink with released extracellular matrix (eg, collagen) from cells cultured on the support. These supports can be formed of a material that can form cross-links with the ECM and/or the cells during tanning. In addition, the structure of the support (for example, porosity, fiber length, fiber density, etc.) can be chosen to allow cross-linking and/or to stimulate cell growth and extracellular matrix release. . The backing is tanned with and to the ECM to form the final product, the fibre-reinforced fabric composite material. Therefore, the backing forms an integral part of the final product, and its dimensions, including thickness, can help determine the final thickness of the resulting leather.

A particular example of a fibrous support that can be used as described herein is silk. Silk (eg, organic and/or synthetic silk) can be formed (eg, spun) to a predetermined fiber thickness and can be used in a woven and/or non-woven sheet that forms the support on which cells can be grown. that release ECM. Such cells can be, for example, skin fibroblasts, smooth muscle cells, etc.

In general, the support is made of a material (such as silk) that can be tanned to cross-link reactive groups on the support (eg, amine and carboxylic acid groups) with the released ECM (eg, collagen).

The fibrous support can provide a large amount of surface area on which cells can be cultured. For example, the backing may be made up of relatively dispersed fibers that form a disperse fibrous backing. A fibrous support can have a lot of surface area to allow for cell growth and to allow for a strong fiber-tissue matrix interface (which can also improve the overall strength of the resulting composite material). In some variations, the length of the fibers that form the support is between 100 nm and 100 µm. In some variations, the length of the fibers is between 100nm and up to 1mm, 10mm, 100mm or 1m. The density of the fibrous material in the support can be between 10 and 100 mg/cc. In some variations, the density of the fibrous material is between 10 and 10,000 mg/cc. The porosity of the fibrous support (including woven/non-woven fibers) can be between 10 and 99%.

In general, it may be advantageous to provide high porosity. This can allow for much cell infiltration and tissue growth, including within the thickness of the support. The support may have any suitable thickness (for example, between 10 |im and 5 mm, for example, between 100 |im and 1 mm, between 100 |im and 500 |im, between 50 |im and 300 |im, etc. , or between any value less than 10 |im, 30 |im, 50 |im, 75 um, 100 um, 200 um, 300 |im, etc. and a value greater than 50 |im, 100 |im, 150 |im , 200 |im, 300 |im, 400 |im, 500 |im, 600 |im, 700 |im, 800 |im, 900 |im, 1mm, 2mm, 3mm, 4mm, 5mm, etc. ). Porosity can be determined as the space (eg, average diameter) between fibers.

In operation, the method of forming the fiber-reinforced tissue composites described herein may include culturing cells on the fibrous support until a desired amount of ECM (eg, collagen) has formed in and/or on the fibrous support. the fibrous support. Optionally, the fibrous support may be formed and/or prepared to receive cells to be cultured thereon. For example, the support can be treated to expose reactive groups or cell attachment sites. In some variations, the fibrous support can be applied in a cell reactor for seeding and growth of cells on the support. As an example, cells, which may be for example a mammalian cell line having known ECM-releasing properties, to be seeded on the prepared fibrous support and allowed to grow, divide and deposit ECM. Cells can be cultured under typical culture conditions for an appropriate period of time (eg, 2-90 days). Additional cells can be added during this period. The resulting material, comprising the fibrous support with the ECM and released cells, can then be called an intermediate (untanned) tissue construct, which can be further processed. This intermediate (untanned) tissue construct may or may not be decellularized (eg, by treatment with alcohols or other agents). The intermediate (non-tanned) tissue construction can then be processed by tanning, which can include traditional tanning chemicals, as well as crosslinking reactions to reinforce the support for the tissue.

An example of various reinforcing and tanning methods that can be used and/or modified is given below. The fibrous support and the cultured ECM grown on the support are tanned (and cross-linked) with each other by this process, and the resulting fiber-reinforced fabric composite materials can be used as an engineered leather material.

A reinforcing step may be included to cross-link the deposited ECM with the ECM-coated fibrous support, followed by a tanning process to produce the look and feel of traditional leather. Although traditional tanning chemicals can effectively cross-link ECM to the support if the support has the appropriate functionality (such as surface amine and carboxylic acid groups), other support surface chemicals and crosslinking agents can be incorporated to strengthen the surface. contact ECM-support. These include epoxy, acrylate, aldehyde, sulfhydryl, diazirines, aryl azides, etc., as well as protected chemical compounds that can be activated upon tissue growth to reduce cytotoxicity of the support surface. The support can be modified with reactive chemicals prior to seeding of cells hanging from spacer molecules of size between 0 Da and 100 MDa. The chemical compounds on the surface of the support can be reacted directly with the ECM or through crosslinking agents with functionality between 1 and 2000 and with a size between 1 Da and 100 MDa. Furthermore cross-linking molecules can be polymerized within the support-ECM construct with a size between 1 Da and 100 MDa. The polymerization of these cross-linking agents can be initiated after tissue growth with triggering factors such as light exposure, temperature change, addition of chemical initiators, etc.

In general, any suitable tanning method can be used, including a method derived from traditional tanning, which can result in leather having the look and feel of traditional tanned hides. For example, after culturing the cells on the supports as described above (eg, in a sterile environment), the intermediate (untanned) tissue construct consisting of cells and ECM grown on the flat fibrous support (eg, lamina relatively thin, but long and wide) can be washed (for example, to remove the growing medium), and tanned. Traditional steps such as lime may be included (for example, treatment with a basic compound such as milk of lime and/or the addition of "activators" including disulfide reducing agents, such as sodium sulfide, cyanides, amines, etc.) . However, such steps can be modified since the intermediate construction does not include hair, nails, and other keratinous matter normally found in native hides. Such steps may be maintained or modified to remove some ECM materials and/or to swell and split the fibers in the intermediate construction or to otherwise prepare collagen in the tanning construction.

The methods may include or avoid (as unnecessary) the use of depilating agents such as sodium sulfide, sodium hydroxide, sodium hydrosulfite, calcium hydrosulfide, dimethylamine, and sodium hydrosulfide.

A step that removes cellular material while preserving the ECM may also be included. Decellularization methods used in tissue engineering can be used for this purpose, including the use of surfactants, enzymes, ultrasonic energy, freeze-thaw cycling, etc.

A deliming step may also be included. For example, the pH of collagen can be lowered to a lower level so that enzymes can act on it. Depending on the end use of the leather, the intermediate construction may be treated with enzymes to soften it, a process called rendering. If tanning is used, once completed, the intermediate tissue construct can be treated first with salt and then with sulfuric acid, if mineral tanning is to be done, which can reduce the pH of collagen to a very low level to facilitate penetration of the mineral tanning agent into the substance. This process is known as pickling. Salt (eg sodium chloride) can penetrate faster than acid and limit the deleterious effect of a sudden drop in pH. If vegetable tanning is used, the tanning agent may be a tannin. Tannins are a class of polyphenolic astringent chemicals found naturally in the bark and leaves of many plants. Tannins bind to collagen proteins and can hide and coat them, making them less soluble in water and more resistant to bacterial attack. The process can also make the material more flexible. Traditionally, the primary barks, processed in bark mills and used today, are chestnut, oak, redor, tanoak, hemlock, quebracho, mangrove, acacia, and myrobalan. Traditionally, the skins are stretched over frames and soaked for several weeks in vats of increasing concentrations of tannin. The intermediate tissue constructs described herein may provide more direct, easier access to tannins and thus may require less processing time overall.

In chrome tanning, before the introduction of the basic chrome species, several steps can be used to prepare the material, as mentioned above, including the introduction of alkaline agents such as sodium hydroxide, restoration of neutral pH, rendering (softening with enzymes) and pickling (lowering the pH of the material being processed, for example, with salt and sulfuric acid).

In traditional tanning, the pH is very acidic when chrome is introduced, to ensure that the chrome complexes are small enough to fit between the fibers and collagen debris. Once desired level of chromium penetration into the substance is achieved, the pH of the material is raised again to facilitate the process. This stage is known as basification. Chrome tanning is usually faster than vegetable tanning.

Chromium(III) sulfate ([Cr(H2O)6]2(SO4)3) has long been considered the most efficient and effective tanning agent. Chromium(III) compounds of the type used in tanning are significantly less toxic than hexavalent chromium. Chromium(III) sulfate dissolves to give the hexaaquachrome(III) cation, [Cr(H ² O) ⁶ ]3+, which at higher pH undergoes processes called olation to give polychrome(III) compounds that are active in tanning, which is the cross-linking of collagen subunits. The chemistry of [Cr(H ² O) ⁶ ]3+ is complex due to the presence of a variety of ligands. Some ligands include the sulfate anion, collagen carboxyl groups, amino acid side chain amine groups, and masking agents. Masking agents are carboxylic acids, such as acetic acid, used to suppress the formation of polychrome (III) chains. Masking agents allow the tanner to further increase the pH to increase collagen reactivity without inhibiting the penetration of chromium(III) complexes.

As mentioned above, collagen is characterized by a high content of glycine, proline and hydroxyproline, usually in the -Gly-Pro-Hypro-Gly- repeat. These residues give rise to the helical structure of collagen. The high collagen content of hydroxyproline allows significant cross-linking by hydrogen bonding within the helical structure. Ionized carboxyl groups (RCO2-) are formed by hydrolysis of collagen by the action of hydroxide. This conversion occurs during the lime process, before the introduction of the tanning agent (chrome salts). The ionized carboxyl groups coordinate as ligands to the chromium(III) centers of the oxo-hydroxide clusters.

Tanning increases the separation between protein chains in collagen from 10 to 17 A. The difference is consistent with crosslinking by polychrome species, the type that arises from olation and oxolation.

After the application of the chrome agent, the bath can be treated with sodium bicarbonate to increase the pH to 4.0-4.3. This increase induces crosslinking between chromium and collagen. The pH increase can normally be accompanied by a gradual increase in temperature up to 40°C. Chrome tanned leather may contain between 4 and 5% chrome. This effectiveness is characterized by its increased hydrothermal stability of the leather, and its resistance to shrinkage in hot water.

Other forms of tanning may be used, including those based on alum, zirconium, titanium, iron salts, or a combination thereof. Blank tanning can be used in the intermediate tissue constructions described herein. Tanning is a method that uses alum and aluminum salts, usually together with other products such as egg yolk, flour and other salts. The material is white tanned by immersing it in a lukewarm solution of potash alum and salts (or equivalent), between 20 and 30°C. the process can increase the flexibility, stretchability, softness and resulting quality of the leather. The addition of egg yolk and flour (or equivalent) to the conventional soaking solution can further enhance its fine handling characteristics. The intermediate tissue construct is then dried and allowed to stabilize.

Depending on the desired finish, the material may be waxed, rolled, lubricated, oiled (eg oil injected), and/or dried. They can be formed suede, nubuck, etc., for example, by inducing surface finishes. The material can be additionally finished by means of a new white tanning. New white tanning agents and/or dyes can be applied to the material to enhance physical strength and desired properties depending on the final product. A final phase, finishing, can be used to apply the finishing material to the surface or to finish the surface.

examples

Figures 10-16 illustrate examples of fiber-reinforced fabric composites and methods for forming them as described above.

For example, Figures 10 and 11 show an example of a fibrous tissue support that can be used. In this example, the support is made of silk fibers. The support has a thickness of approximately 0.5 mm, and fiber diameters of approximately 15-20 microns (eg, as average/mean). In this example, the total density of the material is approximately 80 mg/cc, which corresponds to a porosity of approximately 95%. The orientation of the fibers can affect the overall properties of the materials, and the bonding process can be varied to produce a variety of nonwoven, woven, and knitted architectures. In addition, the length of the fibers can affect the general properties of the material, and the length, diameter and/or porosity of the fibers can vary. For example, nanofibers can be used that are from about 10 µm (microns) to 20 µm or more. Figure 11 shows an enlarged view of a region of the support shown in Figure 10.

Figure 12A shows an example of a "dry" fibrous support (silk) before being seeded with cells. In Figure 12B, the support shown in Figure 12A has been seeded with cells (in this example, bovine dermal fibroblasts) at a density of 8.5x106 cells/ml. The support volume was 100 µl/cm2 support (similar to that shown in Figure 10-11). In this example, cells are seeded on top of the support and have been observed to settle (eg, by gravity) onto the porous support. The support can then be turned, flipped, rotated, etc., which can help distribute the cells and/or culture medium throughout the substrate. In practice, it can be beneficial to have a fibrous substrate that is initially more than 50% porous. As mentioned above, in some variations, the support can be modified to enhance cell adhesion. In the example shown in Figure 12B, cells can be cultured for approximately 4 weeks, with regular media change (eg, every few days); conventional tissue culture techniques can be adapted for use with the support; for example, growth factors, or agents to enhance the release and/or type of deposited ECM can be used. In addition, dynamic culture environments such as perfusion or mechanical loading can be used to enhance ECM deposition. In Figures 12A-12B, the support has a thickness of about 0.5 mm. Thicker media (eg up to 5 cm) can also be used. After four weeks of culture, the fibers of the support are surrounded by tissue (including collagen).

Figure 13 shows a section taken of the exemplary intermediate tissue construction (untanned) shown in Figure 12B in cross-section through the tissue. In this example, the stain shows (in red in the original color version) collagen; the fibers of the support are indicated (in section) by the arrowheads. The sample is shown to have a dense network of collagen extending around and through (eg, within the porous fibrous support). This sample was fixed for histology. Provided in Figure 14 is a scanning electron microscopy image of an intermediate (untanned) tissue construct similar to that shown in Figure 12B.

In Figure 14, a section through a fibrous support on which collagen-releasing cells have been cultured is shown. The fibrous support in this example is silk, and the SEM shows collagen-rich tissue growing throughout the silk fiber support.

Figure 15 shows a comparison between a fibrous support that allows (eg silk) and that does not allow (eg poly(lactic acid), PLLA) reinforcing crosslinking during a tanning process as described herein. document. In Figure 15, cells have been equivalently cultured on four different fibrous supports and tanned: silk 501, high density PLLA 503, polyester 505 and low density PLLA 507. Only the silk support included amine and carboxylic acid groups which are cross-linked with fabric/ECM during the tanning process (chromium salt tanning). Each of these supports are otherwise approximately equivalent. Each is about 4cm long and about 1.5cm wide; silk 501 and low density PLLA 507 are each 0.5mm thick and high density PLLA and polyester are each about 1mm thick. After tanning, only samples where the backing was crosslinkable during tanning (eg, 501 silk) resulted in materials having a texture and appearance similar to native leather. Tanned silk without cultured cells does not; the resulting material (silk alone) is separated.

Figures 16 and 17 illustrate an example of fiber-reinforced fabric composites formed as described herein after tanning. Figure 16 shows an outer surface of a tanned material that has been formed and processed as described above. In this example, the outer leather-like surface has a similar feel to native leather. The material consists of a fibrous silk support on which cells (dermal fibroblasts) have been cultured and the resulting intermediate tissue construction tanned. Figure 17 shows an enlarged view of an edge region of a tanned fiber-reinforced tissue construction (silk). After tanning, a weave gradient developed towards the edge of the silk backing, revealing the silk fibers dispersed throughout the weave matrix (on the right in the figure).

In general, the resulting product will look and feel similar to native leather, but is detectably different (eg, upon examination of the ultrastructure). For example, the resulting material will have the fibrous support material dispersed throughout, and is usually surrounded by ECM (eg collagen); the collagen and the support are tanned and cross-linked with each other. There will normally be an outer layer of fabric on the backing.

For example, described herein are methods for forming a fiber-reinforced biological tissue composite material, the method comprising: culturing cells on a fibrous support until the fibers of the fibrous support are surrounded by tissue comprising extracellular matrix, forming a intermediate fabric construction; and tanning the intermediate fabric construction to crosslink the fibers of the fibrous framework with the fabric. The culture may comprise culturing cells on a fibrous silk support, culturing cells on a fibrous protein support, etc. The fibrous protein support may comprise one or more of: a fibrous wool support, a fibrous silk support, or a fibrous collagen support.

The culture may comprise culturing cells on a protein fiber support comprising amine, carboxylic acid and sulfhydryl groups. The cultivation may comprise culturing cells on a fibrous cellulosic support. The fibrous cellulosic backing may comprise one or more of a fibrous linen backing, a fibrous pineapple backing, or a fibrous cotton backing. Culturing may comprise culturing cells on a synthetic fiber support with cross-linkable surface chemistry.

The fibrous synthetic backing may comprise one or more of a carbon fiber backing, a Kevlar backing, or a fiberglass backing.

Culturing may include culturing cells on the fibrous support until the fibers are surrounded by collagen. The cultivation may include culturing cells on the fibrous support comprising fibers that are between about 100nm and 1m in length. The cultivation may include culturing cells on the fibrous support having a porosity of between 10 and 99%. The culture may comprise culturing mammalian cells on the fibrous support. Tanning can include cross-linking amine, hydroxyl, sulfhydryl, tyrosyl, and carboxylic acid groups on extracellular matrix proteins in the supported fabric. Tanning may comprise cross-linking the fibers of the fibrous support to the fabric through covalent bonds. Tanning may comprise cross-linking the fibers of the fibrous support with the fabric through ionic, hydrophobic and van der Waals interactions.

A fiber-reinforced biological tissue composite may include: a fibrous support comprising a plurality of fibers, wherein the fibers are surrounded by extracellular matrix, wherein the extracellular matrix and the plurality of fibers are crosslinked with each other.

The support may be a naturally occurring protein fiber containing amine hydroxyl, sulfhydryl, tyrosyl and carboxylic acid groups. The support may be a naturally occurring cellulosic fiber containing amine, hydroxyl and carboxylic acid groups. The support may be a synthetic fiber containing surface chemicals to crosslink with the ECM. The fibers can be chemically modified to enhance crosslinking of the fabric with the support.

The support may be chemically modified to contain tissue crosslinking groups including one or more of: amines, carboxylic acids, sulfates, aldehydes, hydrazides, sulfhydryls, diazirines, aryl azides, acrylates, and epoxides.

Tissue crosslinking groups can be protected during tissue growth and activated for crosslinking when tissue growth is complete. The fabric crosslinking groups can hang from the support with a spacer between 1 dalton and 100 megadaltons.

The support can crosslink with the tissue through non-covalent interactions, including ionic, hydrophobic, and van der Waals interactions. The support can be cross-linked with the fabric through covalent bonds. The support can directly react with amine, hydroxyl, sulfhydryl or carboxylic acid groups on the fabric. The support can be reacted with a crosslinking agent that reacts with amine, hydroxyl, sulfhydryl, or carboxylic acid groups on the fabric. The molecular weight of the crosslinking agent can be between 1 dalton and 100 megadaltons. The support can be reacted with amine, hydroxyl, sulfhydryl, or carboxylic acid groups on the fabric through a polymerization reaction that creates polymeric crosslinking agents. The support may comprise woven, knitted or non-woven fibers having a length between 100nm and 1m. The density of the fibrous material in the support can be between 10 and 10,000 mg/cc. The porosity of the fibrous support can be between 10 and 99%.

When a feature or element is referred to herein as "on top" of another feature or element, it may be directly on top of the other feature or element or intermediate features and/or elements may also be present. In contrast, when a feature or element is referred to as “directly above” another feature or element, no intermediate features or elements are present. It will also be understood that, when one feature or element is referred to as "connected", "attached" or "attached" to another feature or element, it may be directly connected, attached or attached to the other feature or element or may be present features or intermediate elements. In contrast, when a feature or element is referred to as "directly connected", "directly attached" or "directly coupled" to another feature or element, no intermediate features or elements are present. Those skilled in the art will also appreciate that references to a structure or feature being disposed "adjacent" to another feature may have portions that overlap or underlie the adjacent feature.

As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprise" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements and/or components, but do not exclude the presence or addition of one or more other characteristics, stages, operations, elements, components and/or groups of the themselves. As used herein, the term "and/or" includes any and all combinations of one or more of the associated enumerated elements and may be abbreviated as "/".

Spatially relative terms, such as "under," "below," "below," "above," "above," and the like, may be used herein for ease of description to describe the relationship of an element or feature to other element(s) or characteristic(s), as illustrated in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of the device or operation in use in addition to the orientation depicted in the figures. For example, if a device is inverted in the figures, elements described as "under" or "below" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "low" can encompass both an over and under orientation. The device may be oriented in another way (rotated 90 degrees or in other orientations) and the spatially relative descriptors used herein may be interpreted accordingly. Similarly, the terms "upward", "downward", "vertical", "horizontal" and the like are used herein for explanatory purposes only, unless specifically indicated otherwise.

Although the terms "first" and "second" may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be referred to as a second feature/element, and similarly, a second feature/element discussed below could be referred to as a first feature/element without departing from the teachings of the present invention.

Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprising", and variations such as "comprising" and "comprising" mean that various components may be used together in methods and articles (eg compositions and apparatus including device and methods). For example, the term "comprising" will be understood to imply the inclusion of any stated step or element, but not the exclusion of any other step or element.

As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if preceded by the word "approximately" or “around,” although the term does not appear expressly. The expression "approximately" or "about" may be used when describing magnitude and/or position to indicate that the described value and/or position are within a reasonable expected range of values and/or positions. For example, a numeric value can have a value that is /- 0.1% of the set value (or range of values), /- 1% of the set value (or range of values), /- 2% of the set value (or range of values), /- 5% of the set value (or range of values), /- 10% of the set value (or range of values), etc. Any numerical value provided herein is also to be understood to include about or about that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "approximately 10" is also disclosed. Any numerical range cited herein is intended to include all subranges included therein. It is also understood that when a value is disclosed that is "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled person. in technique. For example, if the value "X" is disclosed, "less than or equal to X" is also disclosed as well as "greater than or equal to X" (eg, when X is a numeric value). It is also understood that throughout the application, the data is provided in a number of different formats, and that this data represents end points and start points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than greater than or equal to, less than, less than are also considered to be disclosed. or equal to, and equal to 10 and 15 between 10 and 15. It will also be understood that each unit between two particular units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Claims (12)

1. Fiber-reinforced biological fabric composite material, the material comprising: a fibrous support comprising a plurality of fibers, in which the fibers are surrounded by extracellular matrix, and wherein the extracellular matrix and the plurality of fibers are crosslinked with each other. 2. Composite material of biological tissue reinforced with fibers according to claim 1, in which each of the fibers of the plurality of fibers has a length in a range of from 100 nm to 1 m, more preferably from 100 nm to 1 mm . The fiber-reinforced biological tissue composite material according to claim 1, wherein the fibers of the plurality of fibers comprise at least one of woven fibers, knitted fibers or non-woven fibers. 4. Composite material of biological tissue reinforced with fibers according to claim 1, in which the fibrous support has a porosity in a range of from 10% to 99%. The fiber-reinforced biological tissue composite material according to claim 1, wherein the fibrous support has a density within a range of 10 mg/cc to 10,000 mg/cc. 6. Composite material of biological tissue reinforced with fibers according to claim 1, in which the fibrous support has a thickness in a range from 10 µm to 5 mm. The fiber-reinforced biological tissue composite material according to claim 1, wherein the fibrous support comprises a material selected from the group consisting of carbon fiber, Kevlar, fiberglass, a fibrous cellulosic material, fibrous wool, fibrous silk and fibrous collagen. The fiber-reinforced biological tissue composite material according to claim 1, wherein the fibrous support comprises a naturally occurring protein fiber, the naturally occurring protein fiber comprising at least one of a group amine hydroxyl, a sulfhydryl group, a tyrosyl group or a carboxylic acid group. The fiber-reinforced biological tissue composite material of claim 1, wherein the fibrous support has a surface chemistry configured to allow crosslinking between the fibers within the plurality of fibers and the extracellular matrix. 10. The fiber-reinforced biological tissue composite according to claim 1, wherein the fibers of the plurality of fibers are chemically modified to enhance cross-linking of the tissue with the fibrous support. The fiber-reinforced biological tissue composite material of claim 1, wherein the fibrous support is chemically modified to contain a tissue crosslinking group selected from the group consisting of amines, carboxylic acids, sulfates, aldehydes, hydrazides, sulfhydryls , diazirines, aryl-azides, acrylates and epoxides. 12. The fiber-reinforced biological tissue composite material according to claim 1, wherein the extracellular matrix and the plurality of fibers are crosslinked with each other through at least one of covalent bonds, ionic bonds, hydrophobic interactions or van der interactions. waals.